Dr. Alexander Blinov
Head of Cell Biology Laboratory
Institute of Cytology and Genetics
prospect Lavrentjeva, 10
Novosibirsk 630090
Russia
Fax (3832) 35-65-58
Phone: (3832) 35-46-70
We are looking forward to establish collaboration with scientists
interested in study of regulation of expression and transposition of
non-LTR retrotransposons. As a first step we would like to get a
JOINT GRANT with somebody interested in such kind of investigations.
Here is a first draft of a project we propose:
EXPRESSION OF THE NLRCTH1 ON THE DIFFERENT DEVELOPMENTAL STAGES AND
UNDER DIFFERENT PHYSIOLOGICAL CONDITIONS.
We have previously described and characterized two non-LTR
retransposons from the Diptera Chironomus thummi (Blinov et al.,
1993) and C.tentans
(Blinov v et al., 1997). Also we have shown that the distribution of
this elements is restricted by Chironomus genus. It has been shown
that mininimum of three different non-LTR retrotransposons are
present in the Chironomus genus. All of them contain similar
nucleotide sequences in the region of the ORF2 which encodes reverse
transcriptase. We disignated the retrotransposon as NLRCth1.
It has been shown that a transposition of non-LTR retrotransposons
causes haemophilia and adenocarcinoma in man (Kazazian et al, 1988;
Morse et al, 1988), and hybrid dysgenesis in drosophila
(Finnegan,1989). Expression of these elements is particularly high
in certain embryonal carcinoma and teratocarcinoma cell lines
(Leibold et al, 1990). It leads to speculation that expression and
transposition of non-LTR retrotransposons may occur only in certain
developmental stages. Moreover, the synthesis of full-length
transcripts has been shown only for several organisms. We would like
to investigate expression of the NLRCth1 on the different larval
stages of Chironomus thummi using the Nothern blot analysis. The
Chironomus is a very good subject for such kind of investigation.
These animals have several very good separated developmental stages.
Also, it will be interesting to show the influence of some kinds of
stress on the expression of this element since the role of
environmental conditions in transposition has been suggested. Heat
stress, for example, can be used as an inductor for this purpose.
Some transcription factors have been described wich bind specifically
to non-LTR retrotransposon promoter region (Mathias and Scott, 1993).
Such proteins are candidates for factors controlling expression of
retrotransposons. Gene mobility shift assays may be used for
investigations of differential expression of NLRCth1.
The next experimental procedures in this project include:
1. Isolation of nuclear extracts from different stages of Chirnomus
thummi.
2. Constructing hybrid plasmids containing different parts of NLRCth1
promoter region.
3. Gel mobility shift assays analysis, using the nuclear extracts and
DNA probes.
4. DNAse I protection assays.
5. Constructing of Chironomus thummi expression library.
6. Screening of this library using South-West analysis.
7. Isolation and characterization of positive clones.
Using this approach we hope to get answers for two important
questions:
1. Is the expression of non-LTR retrotransposons stage- and
tissue-specific; and whether physiological conditions influence their
expression or not.
2. Whether transcription factors which may control expression of the
NLRCth1 present or not.
REFERENCES
Blinov A.G., Sobanov Y.V., Bogachev S.S., Donchenko A.P., Filippova
M.A. (1993) The Chironomus thummi genome contains a non-LTR
retrotransposon. Mol Gen Genet 237: 412-420.
Blinov A.G., Sobanov Y.V, Scherbik S.V. and Aimanova K.G. (1997) The
Chironomus (Camptochironomus) tentans genome contains two non-LTR
retrotransposons. Genome, in press. (Genome, Febriary 1997)
Finnegan D.J. (1989) Eucariotic transposable elements and genome
evolution. Trends in Genet 5: 103-107.
Kazazian H.H., Wong C., Youssufian H., Scott A.F., Philips D.G.,
Antonarakis S.E. (1988) Haemophilia A resulting from de novo
insertion of L1 sequences represents a novel mechanism for mutation
in man. Nature 332:164-166.
Leibold D.M., Swergold G.D., Singer M.F., Thayer B.A., Dombroski
,B.A., Fanning T.G. (1990) Translation of LINE-1 elements in vitro
and in human cells. Proc Natl Acad Sci USA 87: 6990-6994.
Mathias S.L., Scott A.F. (1993) Promoter binding proteins of an
active human L1 retrotransposon. Bioch Bioph Res Commun 191: 625-632.
Morse B., Rotherg P.G., South V.J., Spandorfer J.M., Astrin S.M.
(1988) Insertional mutagenesis of the myc locus by a LINE-1 sequence
in human breast carcinoma. Nature 333: 87-89.